Untargeted Metabolomics Approach for the Discovery of Environment-Related Pyran-2-ones Chemodiversity in a Marine-Sourced Penicillium restrictum.

Very little is known about chemical interactions between fungi and their mollusc host within marine environments. Here, we investigated the metabolome of a Penicillium restrictum MMS417 strain isolated from the blue mussel Mytilus edulis collected on the Loire estuary, France. Following the OSMAC approach with the use of 14 culture media, the effect of salinity and of a mussel-derived medium on the metabolic expression were analysed using HPLC-UV/DAD-HRMS/MS. An untargeted metabolomics study was performed using principal component analysis (PCA), orthogonal projection to latent structure discriminant analysis (O-PLSDA) and molecular networking (MN). It highlighted some compounds belonging to sterols, macrolides and pyran-2-ones, which were specifically induced in marine conditions. In particular, a high chemical diversity of pyran-2-ones was found to be related to the presence of mussel extract in the culture medium. Mass spectrometry (MS)- and UV-guided purification resulted in the isolation of five new natural fungal pyran-2-one derivatives-5,6-dihydro-6S-hydroxymethyl-4-methoxy-2H-pyran-2-one (1), (6S, 1'R, 2'S)-LL-P880ß (3), 5,6-dihydro-4-methoxy-6S-(1'S, 2'S-dihydroxy pent-3'(E)-enyl)-2H-pyran-2-one (4), 4-methoxy-6-(1'R, 2'S-dihydroxy pent-3'(E)-enyl)-2H-pyran-2-one (6) and 4-methoxy-2H-pyran-2-one (7)-together with the known (6S, 1'S, 2'S)-LL-P880ß (2), (1'R, 2'S)-LL-P880γ (5), 5,6-dihydro-4-methoxy-2H-pyran-2-one (8), (6S, 1'S, 2'R)-LL-P880ß (9), (6S, 1'S)-pestalotin (10), 1'R-dehydropestalotin (11) and 6-pentyl-4-methoxy-2H-pyran-2-one (12) from the mussel-derived culture medium extract. The structures of 1-12 were determined by 1D- and 2D-MMR experiments as well as high-resolution tandem MS, ECD and DP4 calculations. Some of these compounds were evaluated for their cytotoxic, antibacterial, antileishmanial and in-silico PTP1B inhibitory activities. These results illustrate the utility in using host-derived media for the discovery of new natural products.

Exosomes as New Biomarkers and Drug Delivery Tools for the Prevention and Treatment of Various Diseases: Current Perspectives.

Exosomes are nano-sized vesicles secreted by most cells that contain a variety of biological molecules, such as lipids, proteins and nucleic acids. They have been recognized as important mediators for long-distance cell-to-cell communication and are involved in a variety of biological processes. Exosomes have unique advantages, positioning them as highly effective drug delivery tools and providing a distinct means of delivering various therapeutic agents to target cells. In addition, as a new clinical diagnostic biomarker, exosomes play an important role in many aspects of human health and disease, including endocrinology, inflammation, cancer, and cardiovascular disease. In this review, we summarize the development of exosome-based drug delivery tools and the validation of novel biomarkers, and illustrate the role of exosomes as therapeutic targets in the prevention and treatment of various diseases.

New Quinoxaline Derivatives as Dual Pim-1/2 Kinase Inhibitors: Design, Synthesis and Biological Evaluation.

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure-activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

Dibenzofuran Derivatives Inspired from Cercosporamide as Dual Inhibitors of Pim and CLK1 Kinases.

Pim kinases (proviral integration site for Moloney murine leukemia virus kinases) are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Thus, Pim kinases are validated as targets for antitumor therapy. In this context, our combined efforts in natural product-inspired library generation and screening furnished very promising dibenzo[b,d]furan derivatives derived from cercosporamide. Among them, lead compound 44 was highlighted as a potent Pim-1/2 kinases inhibitor with an additional nanomolar IC50 value against CLK1 (cdc2-like kinases 1) and displayed a low micromolar anticancer potency towards the MV4-11 (AML) cell line, expressing high endogenous levels of Pim-1/2 kinases. The design, synthesis, structure-activity relationship, and docking studies are reported herein and supported by enzyme, cellular assays, and Galleria mellonella larvae testing for acute toxicity.

Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis.

BACKGROUND: Azoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis. OBJECTIVES: We investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis. METHODS: Antifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate. RESULTS: Compared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole. CONCLUSIONS: Our data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.

4-cholesten-3-one decreases breast cancer cell viability and alters membrane raft-localized EGFR expression by reducing lipogenesis and enhancing LXR-dependent cholesterol transporters.

BACKGROUND: The alteration of lipid metabolism in cancer cells is recognized as one of the most important metabolic hallmarks of cancer. Membrane rafts defined as plasma membrane microdomains enriched in cholesterol and sphingolipids serve as platforms for signaling regulation in cancer. The main purpose of this study was to evaluate the effect of the cholesterol metabolite, 4-cholesten-3-one, on lipid metabolism and membrane raft integrity in two breast cancer cell lines, MCF-7 and MDA-MB-231. Its ability to reduce cell viability and migration has also been investigated. METHODS: RT-qPCR was performed to evaluate the expression of enzymes involved in lipogenesis and cholesterol synthesis, and ABCG1 and ABCA1 transporters involved in cholesterol efflux. Its effect on cell viability and migration was studied using the MTT assay, the wound healing assay and the Transwell migration assay, respectively. The effect of 4-cholesten-3-one on membrane rafts integrity was investigated by studying the protein expression of flotillin-2, a membrane raft marker, and raft-enriched EGFR by western blot. RESULTS: Interestingly, we found that 4-cholesten-3-one treatment decreased mRNA expression of different enzymes including ACC1, FASN, SCD1 and HMGCR. We further demonstrated that 4-cholesten-3-one increased the expression of ABCG1 and ABCA1. We also found that 4-cholesten-3-one decreased the viability of MCF-7 and MDA-MB-231 cells. This effect was neutralized after treatment with LXR inverse agonist or after LXRß knockdown by siRNA. As a result, we also demonstrated that 4-cholesten-3-one disrupts membrane rafts and cell migration capacity. CONCLUSION: Our results show that 4-cholesten-3-one exerts promising antitumor activity by altering LXR-dependent lipid metabolism in breast cancer cells without increasing lipogenesis.

The amino acid substitution N136Y in Candida albicans sterol 14alpha-demethylase is involved in fluconazole resistance.

Resistance to fluconazole antifungal is an ongoing impediment to a successful treatment of Candida albicans infections. One of the most prevalent mechanisms leading to azole resistance is genetic alterations of the 14α-demethylase, the target of azole antifungals, through point mutations. Site-directed mutagenesis and molecular modeling of 14α-demethylase rationalize biological data about the role of protein substitutions in the azole treatment failure. In this work, we investigated the role of N136Y substitution by site-directed mutagenesis into Pichia pastoris guided by structural analysis. Single amino acid substitutions were created by site-directed mutagenesis into P. pastoris with C. albicans ERG11 gene as template. In vitro susceptibility of P. pastoris transformants expressing wild-type and mutants to azole compounds was determined by CLSI M27-A2 and spot agar methods. The fluconazole effect on ergosterol biosynthesis was analyzed by gas chromatography-mass spectrometry. By microdilution and spot tests, N136Y transformants showed a reduced in vitro susceptibility to fluconazole compared to wild-type controls. As expected, ergosterol/lanosterol ratios were higher in N136Y transformants compared to the wild-type controls after treatment with fluconazole. Molecular modeling suggests that residue Asn136 located within the first mutation hot spot, could play a role during heme and azole binding. These results provide new insights into the structural basis for 14α-demethylase-azole interaction and could guide the design of novel azole antifungals.

Synthesis of new pyridazino[4,5-b]indol-4-ones and pyridazin-3(2H)-one analogs as DYRK1A inhibitors.

New pyridazino[4,5-b]indol-4-ones and pyridazin-3(2H)-one analogs were synthesized and their inhibitory activities against DYRK1A, CDK5/p25, GSK3α/ß and p110-α isoform of PI3K evaluated using harmine as reference. Both furan-2-yl 10 and pyridin-4-yl 19 from the two different series, exhibited submicromolar IC50 against DYRK1A with no activities against the three other kinases. In addition, compound 10 exhibited antiproliferative activities in the Huh-7, Caco2 and MDA-MB-231 cell lines.

Discovery of (7-aryl-1,5-naphthyridin-2-yl)ureas as dual inhibitors of ERK2 and Aurora B kinases with antiproliferative activity against cancer cells.

A novel series of (7-aryl-1,5-naphthyridin-2-yl)ureas was discovered as dual ERK2 and Aurora B kinases inhibitors. Several analogues were active at micromolar and submicromolar range against ERK2 and Aurora B, associated with very promising antiproliferative activity toward various cancer cell lines. Synthesis, structure activity relationship and docking study are reported. In vitro ADME properties and safety data are also discussed.

Study of N1-alkylation of indoles from the reaction of 2(or 3)-aminoindole-3-(or 2)carbonitriles with DMF-dialkylacetals.

The condensation of 2-aminoindole-3-carbonitriles and their 3-aminoindole-2-carbonitrile isomers with various DMF-dialkoxyacetals was investigated under microwaves. The appearance of reactive and versatile alkoxyiminium species allowed convenient access to indole precursors of building blocks with potential biological activity. The experimental results have been rationalised using DFT calculations of theoretical descriptors based on the electrostatic potential.

Synthesis and in vitro antifungal evaluation of 2-(2,4-difluorophenyl)-1-[(1H-indol-3-ylmethyl)methylamino]-3-(1H-1,2,4-triazol-1-yl)propan-2-ols.

We extended our previous studies based on the design of 1-[(1H-indol-5-ylmethyl)amino]-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents toward the identification of new indol-3-ylmethylamino derivatives. The majority of these compounds exhibited antifungal activity against a Candida albicans strain (minimum inhibitory concentrations ranging from 199.0 to 381.0 ng/mL) suggesting an inhibition of 14α-demethylase by sterol analysis studies, but are weaker inhibitors compared to their indol-5-ylmethylamino analogs.

Tetraspanins: useful multifunction proteins for the possible design and development of small-molecule therapeutic tools.

Tetraspanins constitute a well-conserved superfamily of four-span small membrane proteins (TM4SF), with >30 members in humans, with important roles in numerous mechanisms of cell biology. Moreover, tetraspanins associate with either specific partner proteins or another tetraspanin, generating a network of interactions involved in cell and membrane compartmentalization and having a role in cellular development, proliferation, activation, motility, and membrane fusions. Therefore, tetraspanins are considered regulators of cellular signaling and are often depicted as 'molecular facilitators'. In view of these many physiological functions, it is likely that these molecules are important actors in pathological processes. In this review, we present the main characteristics of this superfamily, providing a more detailed description of some significant representatives and discuss their relevance as potential targets for the design and development of small-molecule therapeutics in different pathologies.

New azole antifungals with a fused triazinone scaffold.

We identified a new series of azole antifungal agents bearing a pyrrolotriazinone scaffold. These compounds exhibited a broad in vitro antifungal activity against pathogenic Candida spp. (fluconazole-susceptible and fluconazole-resistant) and were 10- to 100-fold more active than voriconazole against two Candida albicans isolates with known mechanisms of azole resistance (overexpression of efflux pumps and/or specific point substitutions in the Erg11p/CYP51 enzyme). Our lead compound 12 also displayed promising in vitro antifungal activity against some filamentous fungi such as Aspergillus fumigatus and the zygomycetes Rhizopus oryzae and Mucor circinelloides and an in vivo efficiency against two murine models of lethal systemic infections caused by Candida albicans.

Design of new antifungal agents: synthesis and evaluation of 1-[(1H-indol-5-ylmethyl)amino]-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols.

We previously reported on the design and synthesis of 1-[((hetero)aryl- or piperidinylmethyl)amino]-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols showing various degrees of antifungal activity against Candida albicans and Aspergillus fumigatus strains. Now we have identified a series of 1-[(1H-indol-5-ylmethyl)amino] derivatives which exhibited potent MICs (<65 ng mL(-1)) against C. albicans strain. The synthesis and SAR behind the indole scaffold will be discussed.

Synthesis and structure-activity relationships of 2-phenyl-1-[(pyridinyl- and piperidinylmethyl)amino]-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents.

Continuous efforts on the synthesis and structure-activity relationships (SARs) studies of modified 1-benzylamino-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents, allowed identification of new 1-[(pyridinyl- and piperidinylmethyl)amino] derivatives with MIC(80) values ranging from 1410.0 to 23.0ngmL(-1) on Candidaalbicans. These results confirmed both the importance of pi-pi stacking and hydrogen bonding interactions in the active site of CYP51-C. albicans.

Design, synthesis and evaluation of 3-(imidazol- 1-ylmethyl)indoles as antileishmanial agents. Part II.

A new series of 1-benzyl-3-(imidazol-1-ylmethyl)indoles were synthesized according to a previous 3D-QSAR predictive model and assayed for their antiparasitic activity upon Leishmania mexicana promastigotes. The biological results obtained for these twelve molecules showed an IC(50) ranging from 2.3 to 32 microM and mainly illustrated the importance of the hydrophobic parameter the para-position of the benzyl group. In order to improve the activities of these compounds and to check the potential influence of the electronic parameter on this particular position, a Craig diagram was used to select original electro-donating and lipophilic substituents. Synthesis and biological evaluation of ten new compounds (IC(50) between 2.5 and 5.4 microM) confirmed that only the hydrophobic field is essential for a high level of activity.

Design, synthesis, and evaluation of 1-(N-benzylamino)-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents.

A series of 1-(N-benzylamino)-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols 6a-c, 7a-c, 8a, and 9a were prepared in five steps and evaluated for their antifungal activity. The most active compound 7b was docked into a home-made 3D model of the targeted enzyme confirming the importance of Tyr118, His377, and Ser378 residues in its binding mode.

Synthesis of 3,5-bis(2-indolyl)pyridine and 3-[(2-indolyl)-5-phenyl]pyridine derivatives as CDK inhibitors and cytotoxic agents.

We here report the synthesis and biological evaluation of new 3,5-bis(2-indolyl)pyridine and 3-[(2-indolyl)-5-phenyl]pyridine designed as potential CDK inhibitors. Indole, 5-hydroxyindole, and phenol derivatives were used to generate three substitutions of the pyridine. The resulting skeletons were successively exploited to introduce various dimethylaminoalkyl side chains by Williamson type reactions. The synthesis includes Stille or Suzuki type reactions, which were realized on the 3,5-dibromopyridine. The preparation and the use of stannylindoles in mono or bis cross-coupling reactions were also described and each step was optimized and detailed. Kinase assays were realized and shown that nude compounds 7, 18, and 25 inhibited CDK1 in the 0.3-0.7 micromolar range with a good selectivity over GSK-3. Cytotoxicity against CEM human leukemia cells was evaluated with IC(50) values in the 5-15 micromolar range. Precise structure-activity relationships were delineated. Molecular modeling and docking solutions were proposed to complete the studies and to explain the observed SAR in the CDK assays.

Novel 9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitrile derivatives as dual cyclin-dependent kinase 1 (CDK1)/glycogen synthase kinase-3 (GSK-3) inhibitors: synthesis, biological evaluation and molecular modeling studies.

Continuous efforts in microwave-assisted synthesis and the structure-activity relationships' (SARs) studies of novel modified 9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitriles, allowed identification of new amidine and imidate derivatives as potent and dual CDK1/GSK-3 inhibitors. Combination of lead optimization and molecular modeling studies allowed identification of a dual CDK1/GSK-3 inhibitor (compound 13d) with submicromolar values.

Homology modeling of MT1 and MT2 receptors.

Melatonin is a neurohormone synthesized and secreted mainly during the dark period of the circadian cycle by the pineal gland. It has already been proved to be involved in a number of chronobiological processes, most of them being mediated by its membranar receptors MT1 and MT2. Both are members of the GPCR class and, despite the interest they elicit, their 3D structure is still to be described. Models for both human MT1 and MT2 receptors have been constructed by homology modeling, using the X-ray structure of bovine rhodopsin as template. These models have been evaluated in terms of hydrophobic properties of the helices and refined to take into account the rearrangement of GPCRs necessary for their activation, thus leading to a putative activated model for each subtype.